We continue to use the Atomic Force Microscopy (AFM) and custom- built environmental-chamber to elucidate physical-chemical properties of lipid membrane transformations. The physical- chemical properties of lipid membranes are crucially important to many biological processes such as the invasion of infectious agents, sub-cellular trafficking and the application of therapeutic agents. We have sucessfully elucidated the thermodynamic transition enthalpy, entropy and the number of lipids in a typical domain of 2- dimyristoyl-sn-glycero-phosphocholine (DMPC). AFM images revealed two membrane phases that partitioned at the expected DMPC chain-melting temperature. This is a new and absolutely unique application of the AFM which permits the elucidation of membrane-associated biomolecular events critical to medically-important processes. The study has provided an estimation of minimum lipid domain size which is critical to an understanding of membrane 'rafts'. Work is now in progress to (1) verify the domain size estimate and describe domain growth using lipid mixtures with physical-chemically defined critical points (James Dvorak, Fuyuki Tokumasu, Albert Jin). We have demonstrated by gradient density centrifugation (Eriko Nagao) that there is a redistribution of erythrocyte raft-like structures as a consequence of a Plasmodium falciparum infection. Work is in progress to visualize this redistribution by fluorescence microscopy and, using selective extraction techniques by AFM (Fuyuki Tokumasu, James Dvorak). Mathematical modeling Takayuki Arie and Albert Jin, James Dvorak) is being used to understand the change in 'flicker frequency' that occurs as a consequence of malaria infection of erythrocytes. Thus far, we have shown that there is a change in the 'power spectrum' of the erythrocytes and the major 'modes' of the power spectrum. We have begun a kinetic study of the movement of live GFP-labeled sporozoites through the ear of mice (Yasmine Belkaid, Jose Ribeiro, James Dvorak). Thus far, we found that the number of sporozoites injected into a bite area is variable (from 1 to very large numbers). Using a specially designed temperature stage capable of high resolution light microscopy studies, we were able to demonstrate that we can plot the course of movement of individual sporozoites through the ear. These observations are of interest both in terms of their implications to the consequences of the invasion process well as the possible future use of this data to develop methods to identify specific promotors or inhibitors of invasion. We have identified unique nuclear pore complexes (NPCs) in Trypanosoma cruzi (Junko Shimada, James Dvorak). NPCs control bidirectional macromolecular traffic between the nucleus and cytoplasm. Some NPCs are highly conserved in eukaryotes. However, nothing is known about NPCs in the kinetoplastida, most specifically, T. cruzi. We have demonstrated that host cells but not parasites react with anti-lamin A and C. Anti-NPC recognizes a 46 kD protein in T. cruzi and a 62 kD protein in mammalian host cells. These data suggest that some nuclear pore protein epitopes are shared by the nuclear membranes of T. cruzi. The lack of reactivity in T. cruzi with a lamin-like protein may be due either a lack of a specific epitope or the lack of a lamin homologue.